Organic anion-transporting polypeptides 1a/1b control the hepatic uptake of pravastatin in mice

Organic anion-transporting polypeptides (OATPs) mediate the hepatic uptake of many drugs. Hepatic uptake is crucial for the therapeutic effect of pravastatin, a cholesterol-lowering drug and OATP1A/1B substrate. We aimed to gain empirical insight into the relationship between OATPs and pravastatin pharmacokinetics and toxicity. We therefore compared the distribution and toxicity of pravastatin in wild-type and Oatp1a/1b-null mice. Intestinal absorption of pravastatin was not affected by Oatp1a/1b absence, but systemic plasma exposure (AUC) increased up to 30-fold after oral bolus administration. This increased plasma exposure resulted from reduced hepatic uptake, as evident from 10 to 100-fold lower liver-to-plasma concentration ratios. However, the reductions in liver exposure were far smaller (<2-fold) than the increases in plasma exposure. Reduced pravastatin liver uptake in Oatp1a/1b-null mice was more obvious shortly after intravenous administration, with 8-fold lower biliary pravastatin excretion. Although mice chronically exposed to pravastatin for 60 days evinced little muscular toxicity, Oatp1a/1b-null mice displayed 10-fold higher plasma concentrations and 8-fold lower liver concentrations than wild-type mice. Thus, Oatp1a/1b transporters importantly control the hepatic uptake of pravastatin. Activity-reducing human OATP1B polymorphisms may therefore both reduce pravastatin therapeutic efficacy in the liver and increase systemic toxicity risks, thus compromising its therapeutic index in a two-edged way.     

Organic anion transporting polypeptides in the hepatic uptake of PBDE congeners in mice

BDE47, BDE99 and BDE153 are the predominant polybrominated diphenyl ether (PBDE) congeners detected in humans and can induce drug metabolizing enzymes in the liver. We have previously demonstrated that several human liver organic anion transporting polypeptides (humans: OATPs; rodents: Oatps) can transport PBDE congeners. Mice are commonly used to study the toxicity of chemicals like the PBDE congeners. However, the mechanism of the hepatic PBDE uptake in mice is not known. Therefore, the purpose of the current study was to test the hypothesis that BDE47, BDE99, and BDE153 are substrates of mouse hepatic Oatps (Oatp1a1, Oatp1a4, Oatp1b2, and Oatp2b1). We used Human Embryonic Kidney 293 (HEK293) cells transiently expressing individual Oatps and quantified the uptake of BDE47, BDE99, and BDE153. Oatp1a4, Oatp1b2, and Oatp2b1 transported all three PBDE congeners, whereas Oatp1a1 did transport none. Kinetic studies demonstrated that Oatp1a4 and Oatp1b2 transported BDE47 with the greatest affinity, followed by BDE99 and BDE153. In contrast, Oatp2b1 transported all three PBDE congeners with similar affinities. The importance of hepatic Oatps for the liver accumulation of BDE47 was confirmed using Oatp1a4-, and Oatp1b2-null mice.     

Differential effects of pravastatin on the pharmacokinetics of paroxetine in normal and diabetic rats

1.?Diabetes is often accompanied with depression and hypercholesterolemia. It is possible that paroxetine and pravastatin are co-administered to diabetic patients. The aim of this study was to research the differential effect of pravastatin on plasma exposure of paroxetine in normal and diabetic rats.

2.?Pharmacokinetics of paroxetine was investigated following oral administration of paroxetine with and without pravastatin in normal and diabetic rats. Effects of pravastatin on metabolism, intestinal absorption and hepatic uptake of paroxetine were investigated. Activity and expression of hepatic Oatp1 and Oatp2 were also assessed.

3.?Pravastatin decreased plasma exposure of paroxetine in normal rats, but increased exposure of paroxetine in diabetic rats. Pravastatin neither affected metabolism nor intestinal absorption of paroxetine. Data from hepatocytes demonstrated that hepatic uptake of paroxetine were involved in Oatp1 and Oatp2. Diabetes suppressed Oatp1 activity and expression, but enhanced Oatp2 activity and expression. Pravastatin stimulated Oatp1 but inhibited Oatp2 activity.

4.?We concluded that differential effects of pravastatin on plasma exposure of paroxetine in normal and diabetic rats was partly due to the fact that diabetes suppressed Oatp1 activity and expression but enhanced Oatp2 activity and expression as well as that pravastatin stimulated Oatp1 activity but inhibited Oatp2 activity.     

Organic anion-transporting polypeptide (OATP) 2B1 contributes to the cellular uptake of theaflavin

Organic anion-transporting polypeptide (OATP) 2B1 has been reported in the apical membranes of the human small intestinal epithelium, where it contributes to the intestinal absorption of pharmacologically active drugs. To investigate the potential for OATP2B1-mediated drug–food interactions, the effects of several polyphenolic compounds on OATP2B1-mediated estrone-3-sulfate (E3S) transport were studied by using OATP2B1-expressing HEK293 cells. Our results showed that some compounds, especially theaflavin, were strong inhibitors of OATP2B1-mediated E3S uptake. Theaflavin showed a significantly higher uptake into the OATP2B1-expressing HEK293 cells than the control cells. The concentration dependence of the uptake of theaflavin was determined over a range of concentrations (0.5–100 μM) and the kinetic parameters (K_m and V_max) of theaflavin uptake were found to be 5.12 ± 0.67 μM and 41.6 ± 1.3 pmol/mg protein/min, respectively. The OATP2B1-mediated theaflavin uptake was inhibited by known OATP2B1 substrates such as E3S, bromsulphthalein (BSP), dehydroepiandrosterone-3-sulfate (DHEAS), and fluvastatin. Our results indicate that theaflavin is a novel substrate of OATP2B1. The results of this study might be helpful to predict the potential OATP2B1-mediated drug–theaflavin interactions and to avoid undesirable clinical consequences.     

Functional involvement of organic cation transporter1 (OCT1/Oct1) in the hepatic uptake of organic cations in humans and rats

The contribution of organic cation transporters to the saturable component in the hepatic uptake of 1-methyl-4-phenylpyridinium (MPP), tetraethylammonium (TEA), cimetidine, and metformin was examined by the use of human/rat organic cation transporter (hOCT1/rOct1)-expressing cells and human/rat hepatocytes. Transfection of rOct1 resulted in a considerable increase in the uptake of metformin, whereas that of hOCT1 resulted in only a slight increase. All test compounds (MPP, TEA, cimetidine, and metformin) accumulated in human and rat hepatocytes in a carrier-mediated manner. The K_m values for the uptake of MPP, TEA, cimetidine, and metformin into human and rat hepatocytes were comparable with those into hOCT1 and rOct1-expressing cells, respectively. In addition, the relative uptake activities, which were obtained by normalizing the intrinsic uptake clearances of TEA, cimetidine, and metformin against those values of MPP in human and rat hepatocytes, were similar with the uptake activities in hOCT1 and rOct1, respectively. These results suggest that the saturable component in the hepatic uptake of these cationic compounds may be mediated mainly by hOCT1/rOct1; therefore, it is meaningful to evaluate the saturable uptake profile of cationic compounds by the liver using both hOCT1/rOct1-expressing cells and human/rat hepatocytes.     

The inhibitory effects of five alkaloids on the substrate transport mediated through human organic anion and cation transporters

1.?Human solute carrier transporters (SLCs) are important membrane proteins mediate the cellular transport of many endogenous and exogenous substances. Organic anion/cation transporters (OATs/OCTs) and organic anion transporting polypeptides (OATPs) are essential SLCs involved in drug influx. Drug–drug/herb interactions through competing for specific SLCs often lead to unsatisfied therapeutic outcomes and/or unwanted side effects. In this study, we comprehensively investigated the inhibitory effects of five clinically relevant alkaloids (dendrobine, matrine, oxymatrine, tryptanthrin and chelerythrine) on the substrate transport through several OATs/OCTs and OATPs.

2.?We performed transport functional assay and kinetic analysis on the HEK-293 cells over-expressing each SLC gene.

3.?Our data showed tryptanthrin significantly inhibited the transport activity of OAT3 (IC₅₀?=?0.93?±?0.22?μM, K_i?=?0.43?μM); chelerythrine acted as a potent inhibitor to the substrate transport mediated through OATP1A2 (IC₅₀?=?0.63?±?0.43?μM, K_i?=?0.60?μM), OCT1 (IC₅₀?=?13.60?±?2.81?μM) and OCT2 (IC₅₀?=10.80?±?1.16?μM).

4.?Our study suggested tryptanthrin and chelerythrine could potently impact on the drug transport via specific OATs/OCTs. Therefore, the co-administration of these alkaloids with drugs could have clinical consequences due to drug–drug/herb interactions. Precautions should be warranted in the multi-drug therapies involving these alkaloids.     

A comparison of uptake of metformin and phenformin mediated by hOCT1 in human hepatocytes

Metformin, a biguanide that has been used to treat type 2 diabetes mellitus, is reportedly transported into human hepatocytes by human organic cation transporter 1 (hOCT1). The objective of this study was to investigate differences in the hepatic uptake of metformin and phenformin, a biguanide derivative similar to metformin. Special focus was on the role of active transport into cells. Experiments were therefore performed using human cryopreserved hepatocytes and hOCT1 expressing oocytes. Both biguanides proved to be good substrates for hOCT1. However, phenformin exhibited a much higher affinity and transport activity, with a marked difference in uptake kinetics compared with metformin. Both biguanides were transported actively by hOCT1, with the active transport components much greater than passive transport components in both cases, suggesting that functional changes in hOCT1 might affect the transport of both compounds to the same degree. This study for the first time produced detailed comparative findings for uptake profiles of metformin and phenformin in human hepatocytes and hOCT1 expressing oocytes. It is considered that hOCT1 may not be the only key factor that determines the frequency of metformin and phenformin toxicity, considering the major contribution of this transporter to the total hepatic uptake and comparable width of their therapeutic concentrations. Copyright © 2009 John Wiley & Sons, Ltd.     

伊伐布雷定肝脏转运机制的初步研究

目的：探讨伊伐布雷定肝脏转运与OCT1的相关性。方法：将雄性SD大鼠分为对照组与实验组,对照组伊伐布雷定（1 mg·kg^-1）灌胃给药,实验组OCT1抑制剂雷尼替丁（0.8 mg·kg^-1）和伊伐布雷定（1.0 mg·kg^-1）相继灌胃给药,比较2组间伊伐布雷定的药动学参数;采用原代肝细胞,分为对照组：伊伐布雷定（0.2,0.5,1 μmol·L^-1）;实验组：雷尼替丁（0.5,1.0,2.0 μmol·L^-1）+伊伐布雷定（0.2,0.5,1 μmol·L^-1）,探讨雷尼替丁对伊伐布雷定肝脏摄取的影响。结果：合用雷尼替丁后,伊伐布雷定药动学参数C_max、AUC_0-t、AUC_0-∞分别增加了115.41%,128.73%,128.05%,而CLz/F值降低了56.35%,差异有显著性（P<0.05或P<0.01）。雷尼替丁明显抑制肝细胞对伊伐布雷定的摄取,抑制作用随雷尼替丁的浓度增加而增强。结论：伊伐布雷定肝脏转运很可能与OCT1有关。     

Honey flavonoids inhibit hOATP2B1 and hOATP1A2 transporters and hOATP-mediated rosuvastatin cell uptake in vitro

1. Some flavonoids contained in the common diet have been shown to interact with important membrane uptake transporters, including organic anion transporting polypeptides (OATPs). OATP2B1 and OATP1A2 expressed in the apical membrane of human enterocytes may significantly contribute to the intestinal absorption of drugs, e.g. statins. This study is aimed at an evaluation of the inhibitory potency of selected food honey flavonoids (namely galangin, myricetin, pinocembrin, pinobanksin, chrysin and fisetin) toward hOATP2B1 and hOATP1A2 as well as at examining their effect on the cellular uptake of the known OATP substrate rosuvastatin.

2. Cell lines overexpressing the hOATP2B1 or hOATP1A2 transporter were employed as in vitro model to determine the inhibitory potency of the flavonoids toward the OATPs.

3. Chrysin, galangin and pinocembrin were found to inhibit both hOATP2B1 and hOATP1A2 in lower or comparable concentrations as the known flavonoid OATP inhibitor quercetin. Galangin, chrysin and pinocembrin effectively inhibited rosuvastatin uptake by hOATP2B1 with IC₅₀ ～1–10?μM. The inhibition of the hOATP1A2-mediated transport of rosuvastatin by these flavonoids was weaker.

4. The found data indicate that several of the tested natural compounds could potentially affect drug cellular uptake by hOATP2B1 and/or hOATP1A2 at relative low concentrations, a finding which suggests their potential for food–drug interactions.     

有机阳离子转运体在脑内分布的药理学意义

有机阳离子转运体（OCT）是一类多功能双向转运体,参与有机阳离子药物、毒物以及内源性物质的转运。OCT包括OCT1、OCT2和OCT3这3种转运蛋白,其在肝、肠和肾等外周组织表达量较高,在大脑中表达量较低,但发挥着重要作用。有证据表明,OCT可能在多巴胺转运体、去甲肾上腺素转运体和5-羟色胺转运体等单胺类递质高亲和转运体被阻断后发挥代偿作用;OCT在血脑屏障中表达的改变可能影响某些外源性药物和毒性物质的脑内浓度;OCT介导的药物相互作用可能增加某些药物中枢神经系统毒副作用的风险。综述了OCT在脑内的分布,以及其在脑内内源性物质稳态调控中的作用和在药理学研究中的意义,旨在为进一步研究OCT在脑内药物转运和脑内疾病中的作用提供参考。     

Regulation of Renal Organic Ion Transporters in Cisplatin-Induced Acute Kidney Injury and Uremia in Rats

Purpose  The purpose of this study was to examine the regulation of renal organic ion transporters in cisplatin-induced acute kidney injury (AKI) and its relation with indoxyl sulfate (IS), a uremic toxin. Methods  The IS concentrations in the serum and kidney were monitored by high-performance liquid chromatography. Uptake of p-aminohippuric acid, estrone-3-sulfate and tetraethylammonium were examined using renal slices. Real-time PCR and immunoblotting were performed to examine the mRNA and protein expression of rOATs, rOCTs and rMATE1 in the kidney, respectively. Results  The serum and renal IS levels were markedly elevated in cisplatin-treated rats. However, this effect was largely reversed by administration of AST-120, an oral charcoal adsorbent. The functions of renal basolateral organic anion and cation transporters were reduced in cisplatin-treated rats. The levels of mRNA and protein corresponding to rOAT1, rOAT3, rOCT2 and rMATE1, but not rOCT1, were depressed in the kidney of cisplatin-treated rats. Administration of AST-120 to cisplatin-treated rats partially restored the function and expression level of these transporters. Conclusions  Cisplatin-induced AKI causes down-regulation of renal organic ion transporters accompanied by accumulation of serum and renal IS. IS could be involved in the mechanism of down-regulation of rOAT1, rOAT3 and rMATE1 under cisplatin-induced AKI.     

Organic anion transporting polypeptides and organic cation transporter 1 contribute to the cellular uptake of the flavonoid quercetin

Flavonoids such as quercetin and kaempferol mediate several health protective effects, e.g., anticancer effects. They are inhibitors of organic anion transporting polypeptides (OATP) and organic cation transporters (e.g., OCT2). However, little is known whether such transporters contribute to the cellular uptake of flavonoids. Therefore, we investigated the cellular uptake of kaempferol and quercetin using HEK293 cell lines stably expressing different human OATPs or OCT1. Kaempferol was not a substrate of any of the investigated transporters (OATP1A2, OATP1B1, OATP1B3, OATP2A1, OATP2B1, OATP3A1, OATP4A1, OATP5A1, and OCT1). Quercetin showed a significantly higher uptake into the HEK293-OATP1A2, HEK293-OATP2A1, HEK293-OATP2B1, and HEK293-OCT1 cells compared to control cells. The OATP1A2-, OATP2B1-, and OCT1-mediated quercetin uptake was inhibited by known inhibitors such as naringin, cyclosporin A, and quinidine, respectively. The cellular accumulation of quercetin into HEK293-OATP2A1 cells was not inhibited by prostaglandin E₂ and diclofenac. The ionophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) reduced the net uptake of quercetin by increasing the uptake in the HEK293-control cells and causing no significant change in the HEK293-OATP2B1 cells indicating that quercetin follows the FCCP-driven proton flux through the plasma membrane. In addition to passive diffusion, the SLC transporters OATP1A2, OATP2B1, and OCT1 contribute to cellular accumulation of quercetin.     

Interactions of n-Tetraalkylammonium Compounds and Biguanides with a Human Renal Organic Cation Transporter (hOCT2)

Pharmaceutical Research -     

小檗碱对有机阳离子药物转运体抑制作用研究

目的 研究小檗碱对人有机阳离子转运蛋白（OCTs）—OCT1、OCT2、OCT3、OCTN1和OCTN2的抑制作用。方法 应用由转染试剂Lipo 3 000介导的动物细胞转基因方法、经筛选得到各药物转运体过表达细胞株S2-OCT1、S2-OCT2、S2-OCT3、S2-OCTN1和S2-OCTN2;通过检测OCTs介导的放射性探针底物的跨膜转运,建立OCTs体外评价模型;以野生型（WT）细胞为对照组,应用各转运体抑制剂验证其活性;应用上述方法观察小檗碱对各转运体的抑制作用,并计算小檗碱对各药物转运体活性的半数抑制浓度（IC₅₀）。结果 各转运体细胞组与各自WT细胞株比较,转运活性均提高了5倍多,加入抑制剂后,转运活性均明显下降;小檗碱对OCT1、OCT2、OCT3和OCTN1抑制作用较强,对OCTN2的抑制作用相对较弱,IC₅₀分别为7.63、6.80、2.25、4.66和210.34 μmol/L。结论 小檗碱对这5种有机阳离子转运体均有抑制作用,其中对OCT1、OCT2、OCT3、OCTN1的抑制作用较强,发生由其介导的DDI的可能性较大,对OCTN2的抑制作用相对较弱。     

Jatrorrhizine reduces 5-HT and NE uptake via inhibition of uptake-2 transporters and produces antidepressant-like action in mice

Abstract

1. Jatrorrhizine is an active ingredient found in various traditional Chinese medicinal plants. Based on our previous finding that jatrorrhizine was a potent inhibitor of OCT2 and OCT3, the aim of the present study was to explore whether jatrorrhizine has an antidepressant-like action action via inhibition of uptake-2 transporters.

2. In vitro uptake tests showed that jatrorrhizine strongly inhibited PMAT-mediated MPP⁺ uptake with an IC₅₀ value of 1.05?μM and reduced 5-HT and NE uptake mediated by hOCT2, hOCT3 and hPMAT with IC₅₀ values of 0.1–1?μM (for OCT2 and OCT3) and 1–10?μM (for PMAT).

3. In mouse synaptosomes, jatrorrhizine suppressed 5-HT and NE uptake in a concentration dependently manner, where the role of uptake-2 inhibition is significant.

4. The antidepressant-like action of jatrorrhizine was evaluated by mouse tail suspension test (TST). The TST showed that one week of jatrorrhizine (5, 10 and 20?mg/kg, i.p.) or venlafaxine (20?mg/kg, i.g.) can significantly reduce the duration of immobility when compared with vehicle control group.

5. The concentration of jatrorrhizine shows a dose-dependent increase in brain tissues.

6. Our study suggested that jatrorrhizine might be used as an antidepressant agent via inhibition of uptake-2 transporters.     

Organic anion transporting polypeptides expressed in liver and brain mediate uptake of microcystin

Microcystins are toxins produced by freshwater cyanobacteria. They are cyclic heptapeptides that exhibit hepato- and neurotoxicity. However, the transport systems that mediate uptake of microcystins into hepatocytes and across the blood-brain barrier have not yet been identified. Using the Xenopus laevis oocyte expression system we tested whether members of the organic anion transporting polypeptide superfamily (rodent: Oatps; human: OATPs) are involved in transport of the most common microcystin variant microcystin-LR by measuring uptake of a radiolabeled derivative dihydromicrocystin-LR. Among the tested Oatps/OATPs, rat Oatp1b2, human OATP1B1, human OATP1B3, and human OATP1A2 transported microcystin-LR 2- to 5-fold above water-injected control oocytes. This microcystin-LR transport was inhibited by co-incubation with the known Oatp/OATP substrates taurocholate (TC) and bromosulfophthalein (BSP). Microcystin-LR transport mediated by the human OATPs was further characterized and showed saturability with increasing microcystin-LR concentrations. The apparent K(m) values amounted to 7 +/- 3 microM for OATP1B1, 9 +/- 3 microM for OATP1B3, and 20 +/- 8 microM for OATP1A2. No microcystin-LR transport was observed in oocytes expressing Oatp1a1, Oatp1a4, and OATP2B1. These results may explain some of the observed organ-specific toxicity of microcystin-LR. Oatp1b2, OATP1B1, and OATP1B3 are responsible for microcystin transport into hepatocytes, whereas OATP1A2 mediates microcystin-LR transport across the blood-brain barrier.     

Summarizing studies using constitutive genetic deficiency to investigate behavioural influences of uptake 2 monoamine transporters

Burgeoning literature demonstrates that monoamine transporters with high transport capacity but lower substrate affinity (i.e., uptake 2) contribute meaningfully to regulation of monoamine neurotransmitter signalling. However, studying behavioural influences of uptake 2 is hindered by an absence of selective inhibitors largely free of off-target, confounding effects. This contrasts with study of monoamine transporters with low transport capacity but high substrate affinity (i.e., uptake 1), for which there are many reasonably selective inhibitors. To circumvent this dearth of pharmacological tools for studying uptake 2, researchers have instead employed mice with constitutive genetic deficiency in three separate transporters. By studying baseline behavioural shifts, plus behavioural responses to environmental and pharmacological manipulations—the latter primarily targeting uptake 1—investigators have been creatively characterizing the behavioural, and often sex-specific, influences of uptake 2. This non-systematic mini review summarizes current uptake 2 behaviour literature, highlighting emphases on stress responsivity in organic cation transporter 2 (OCT2) work, psychostimulant responsivity in OCT3 and plasma membrane monoamine transporter (PMAT) investigations, and antidepressant responsivity in all three. Collectively, this small but growing body of work reiterates the necessity for development of selective uptake 2-inhibiting drugs, with reviewed studies suggesting that these might advance personalized treatment approaches.     

Creatinine Transport by Basolateral Organic Cation Transporter hOCT2 in the Human Kidney

PURPOSE: Creatinine is excreted into urine by tubular secretion in addition to glomerular filtration. The purpose of this study was to clarify molecular mechanisms underlying the tubular secretion of creatinine in the human kidney. METHODS: Transport of [14C]creatinine by human organic ion transporters (SLC22A) was assessed by HEK293 cells expressing hOCT1, hOCT2, hOCT2-A, hOAT1, and hOAT3. RESULTS: Among the organic ion transporters examined, only hOCT2 stimulated creatinine uptake when expressed in HEK293 cells. Creatinine uptake by hOCT2 was dependent on the membrane potential. The Michaelis constant (Km) for creatinine transport by hOCT2 was 4.0 mM, suggesting low affinity. Various cationic drugs including cimetidine and trimethoprim, but not anionic drugs, markedly inhibited creatinine uptake by hOCT2. CONCLUSION: These results suggest that hOCT2, but not hOCT1, is responsible for the basolateral membrane transport of creatinine in the human kidney.     

有机阳离子转运体的基因多态性研究进展

人体的肝脏、肾脏和肠道上广泛分布有各种转运蛋白,其中有机阳离子转运体（OCTs）负责转运一些内、外源性的有机阳离子的转运。在此我们主要讨论的是OCTs家族的成员OCT1和OCT2的基因多态性以及其功能意义。同时我们也就OCT对于药物代谢动力学以及药效学的影响进行阐述,以及OCTs在药物相互作用上的意义。     

Xenobiotic transporters of the human organic anion transporting polypeptides (OATP) family

1.?The organic anion transporting polypeptides (humans OATP; other species Oatp) belong to the SLCO gene superfamily of transporters and are twelve transmembrane domain glycoproteins expressed in various epithelial cells. Some OATPs/Oatps are expressed in a single organ, while others are expressed ubiquitously.

2.?The functionally characterized members mediate sodium-independent transport of a variety of structurally independent, mainly amphipathic organic compounds, including bile salts, hormones and their conjugates, toxins, and various drugs.

3.?This review summarizes the general features and the substrates of the eleven human OATPs. Furthermore, it reviews what is known about the mechanism of their multispecificity, their predicted structure, their role in drug–food interactions, and their role in cancer.

4.?Finally, some open questions are raised that need to be addressed to advance OATP research in the near future.